Eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) are ω3-polyunsaturated fatty acids that are metabolically active. A large number of scientific studies have produced data suggesting that EPA and/or DHA are beneficial in the prevention and treatment of a variety of medical conditions, including coronary heart disease, blood platelet aggregation, abnormal cholesterol levels etc.
EPA and/or DHA can be sourced from fish oil, e.g. fish oil from cod liver, herring pilchard, menhaden, tuna, saury fish and mullet. However, fish oil fluctuates in price and quality. Furthermore, there are concerns regarding contamination of fish oil with pesticides and heavy metals. Thus, there is increasing interest for another natural source of the aforementioned ω3-fatty acids, i.e. microalgae.
The extraction of ω3-PUFAs from microalgae poses a major challenge. Typically, bioseparation of ω3-PUFAs from microalgae involves removal of insolubles, isolation of products, purification and polishing. The first step in downstream recovery of polyunsaturated fatty acids (PUFAs) from microalgae is extraction. Extraction should be fast, efficient and gentle in order to reduce degradation of the lipids or fatty acids. As explained by Robles Medina et al. (Biotechnology Advances, Vol. 16, No. 3, (1998), 517-580): “The extraction solvents used should be inexpensive, volatile (for ready removal later), free from toxic or reactive impurities (to avoid reaction with the lipids), able to form a two-phase system with water (to remove non-lipids), and be poor extractors of unwanted components (e.g. proteolipids, small molecules).
Several solvent extraction techniques for isolating ω3-PUFAs from microalgae have been described in the prior art. Frequently, microalgae biomass is subjected to cell disruption before being contacted with the extraction solvent in order to maximize recovery of intracellular products. Cell disruption may be achieved by high-pressure homogenization, agitation in the presence of glass and ceramic beads in bead mills, ultrasonication, chemical lysis or by grinding dried biomass. In commercial processes dried lyophilized microalgae biomass is usually used as a starting material for the solvent extraction process as it produces high extraction yields.
It is known in the art that hexane, chloroform, diethyl ether and ethanol can extract ω3-PUFAs such as EPA and DHA. Apolar solvents such as chloroform, hexane or diethyl ether offer the advantage that non-lipid contaminants hardly dissolve in these in solvents. However, these apolar solvents do not completely extract polar lipids (e.g. phosphatides and glycolipids) because of their limited solubility in these solvents. In order to optimize extraction yields, experiments have been conducted with a variety of solvent mixtures, e.g. hexane/ethanol, hexane-isopropanol and chloroform/methanol/water. Ethanol is capable of extracting ω3-PUFAs from microalgae in relatively high yields. However, ethanol will also extract water and a wide range of polar components. This is why it has been advocated to submit ethanol extracts to another solvent extraction with an apolar solvent or an isolation step (e.g. chromatography) to separate a lipid-enriched fraction.
A. R. Fajardo et al. (Eur. J. Lipid Sci. Tehcnol. 109 (2007) 120-126) describes a method for extracting lipids from microalgae (Phyaeodactylym tricornutum) comprising the following steps:                combining lyophilized biomass with ethanol and stirring for 24 hours at room temperature;        filtration to produce a crude extract;        addition of water and hexane to the crude extract to produce a biphasic system; and        separation of the biphasic system in a hexanic phase and a hydroalcoholic phase.        
Existing methods for isolating Ω3-PUFAs from microalgae suffer from a number of drawbacks. First of all, most if not all of these methods employ apolar solvents such as hexane, chloroform or diethyl ether. The handling of these solvents poses a safety hazard as they are highly explosive and/or toxic. Furthermore, these apolar solvents must be essentially completely removed from the final product (ω3-PUFAs containing oil) as only trace levels of these solvents are allowed in food ingredients.
Another drawback of existing isolation methods resides in their complexity, notably the number of isolation steps employed and/or the need for derivatisation of ω3-PUFAs containing lipids.
EP-A 1 178 118 describes a process for obtaining an oil from microbial cells, the process comprising:
a) disrupting the cell walls of the microbial cells to release the oil; and
b) separating the oil from at least part of the cell wall debris formed in (a).
The examples of this European patent application describe processes in which a fungus (Mortierella alpina) and an alga (Oypthecodinium cohnii) are disrupted by high pressure homogenization, followed by centrifugation which produced an oily top layer and a lower aqueous layer containing the cell debris.